1. Field of the Invention
The present invention relates generally to the fields of cancer therapy and gene therapy. More particularly, it concerns the use of PEA3, including but not limited to human PEA3 (hPEA3), to prevent and treat various transformation events.
2. Description of Related Art
It is well established that a variety of cancers are caused, at least in part, by genetic abnormalities that result in either the over-expression of one or more genes, or the expression of an abnormal or mutant gene or genes. For example, in many cases, the expression of oncogenes is known to result in the development of cancer. xe2x80x9cOncogenesxe2x80x9d are genetically altered genes whose mutated expression product somehow disrupts normal cellular function or control (Spandidos et al., 1989).
Most oncogenes studied to date have been found to be xe2x80x9cactivatedxe2x80x9d as the result of a mutation, often a point mutation, in the coding region of a normal cellular gene, i.e., a xe2x80x9cproto-oncogenexe2x80x9d, that results in amino acid substitutions in the expressed protein product. This altered expression product exhibits an abnormal biological function that takes part in the neoplastic process (Travali et al., 1990). The underlying mutations can arise by various means, such as by chemical mutagenesis or ionizing radiation. A number of oncogenes and oncogene families, including ras, myc, neu, raf, erb, src, fms, jun and abl, have now been identified and characterized to varying degrees (Travali et al., 1990; Bishop, 1987).
The ras gene family of cellular oncogenes encodes small GTP-binding proteins. ras genes have been found mutated in wide variety of human tumors. The ras protein (Ras) is a central component in intracellular signaling pathways involved in the transduction of stimuli that induce growth and/or differentiation. In mammalian cells it is activated by guanine nucleotide releasing factors and in the active state binds and activates the serine/threonine protein kinase encoded by the raf proto-oncogene. raf is involved in intracellular signal transduction of a wide range of stimuli inducing growth and/or differentiation. It can be activated by binding to an activated ras protein, and in turn phosphorylates and activates the protein kinase map.
myc is a cellular oncogene that is involved in the chromosome translocation t(8;13) (q24;q32) found in Burkitt""s lymphoma where it is translocated into the immunoglobulin heavy chain gene. It encodes a transcription factor, forming a DNA-binding hetero-oligomer with the transcription factor Max.
Other cellular genes which can act as oncogenes when mutated include the tyrosine protein kinases src and fms; erb the gene encoding the epidermal growth factor receptor; fos whose product (Jun) in a complex with the product of the fos gene (Fos) forms the activating transcription factor AP-1; and abl whose mutation is characterized by the t(9;22)q34;q11) translocation in patients with chronic myeloid leukemia to generate a composite gene comprised of exons from the BCR locus on chromosome 22 and the abl gene on chromosome 9.
The neu gene (also known as HER2/neu or c-erb-2) encodes a 185-kDa transmembrane tyrosine kinase (p185neu) with homology to epidermal growth factor receptor (Hung et al., 1986; Coussens et al., 1985; Schechter et al., 1984; Sanba et al., 1985; Yamamoto et al., 1986). Enhanced expression of neu is known to be involved in many human cancers, including non-small cell lung cancers (NSCLC) and has been shown to correlate with poor patient survival in NSCLC (Kern et al., 1990; Schneider et al., 1981; Weiner et al., 1990). Cellular and animal studies have shown that an increase in neu tyrosine kinase activity increases the expression of malignant phenotypes (Muller et al., 1988; Hudziak et al., 1987; Muthuswamy et al., 1994; Yu et al., 1991; Yu et al., 1993; Hung et al., 1989; Sistonen et al., 1989; Yu et al., 1994).
The neu oncogene, was first identified in transfection studies in which NIH 3T3 cells were transfected with DNA from chemically induced rat neuroglioblastomas (Shih et al., 1981). The p185 protein encoded by neu has an extracellular, transmembrane, and intracellular domain, and therefore has a structure consistent with that of a growth factor receptor (Schechter et al., 1984). The human neu gene was first isolated due to its homology with v-erbB and EGF-r probes (Senba et al., 1985).
The neu oncogene plays an important role in carcinogenesis, for example, the gene is amplified in approximately 20-30% of human breast cancer. Amplified expressions of the neu oncogene in transfected 3T3 cells induces malignant transformation. neu expression has also been detected in ovarian cancer and its overexpression results in poor prognosis. The expression of neu oncogenes in human tumor cells induce resistance to several host cytotoxic mechanisms.
Along with an increased proliferative potential, neu-mediated cancers appear to be resistant to host defense mechanisms. Studies have shown that overexpression of the neu oncogene in transfected cells results in resistance to tumor necrosis factor, a major effector molecule in macrophage-mediated tumor cell cytotoxicity.
Thus, neu oncogene expression is correlated with the incidence of cancers of the human breast and female genital tract. Moreover, amplification/overexpression of this gene has been directly correlated with relapse and survival in human breast cancer (Slamon et al., 1987; 1989). It is important to evolve information regarding the neu oncogene, particularly information that could be applied to reversing or suppressing the oncogenic progression that seems to be elicited by the presence or activation of this gene. Unfortunately, little has been previously known about the manner in which one may proceed to suppress the oncogenic phenotype associated with the presence of oncogenes such as the neu oncogene.
In addition, neu overexpression in NSCLC is associated with shortened survival. In vitro experimental models have provided evidence that, in the murine cell NIH 3T3, oncogenes increase drug resistance. Tsai et al., 1993 and 1995 used a NSCLC model to demonstrate that activation of an oncogene is quantitatively associated with intrinsic chemoresistance in human malignant cells. This resistance is observed with a variety of drugs that are structurally unrelated and act on different targets and/or by different mechanisms. Thus increased expression of neu oncogene enhances chemoresistance to a wide variety of chemotherapeutic agents (Tsai, 1993) including cisplatin, doxorubicin, and VP16 (Tsai et al., 1993; Tsai et al., 1995). The association of neu overexpression in cancer cells with malignant phenotypes and chemoresistance provides a plausible interpretation for the poor clinical outcome for patients with neu-overexpressing tumors.
Although breast cancer diagnosed in its earliest clinical stages (stage 0, stage Ia) is highly curable, the cure rate for more advanced stages drops precipitously, even after modem combined-modality treatments. Metastatic breast cancer responds to both chemotherapy and hormone therapy, and most patients can be palliated adequately during the 1 to 3 years of usual survival. However, metastatic breast cancer is considered incurable, as demonstrated by the relentless death rates, regardless of the treatment modality utilized. Front-line chemotherapy or hormone therapy programs for correctly selected patients produce objective responses in 50% to 70% of patients, but the median duration of response is usually less than one year. Response rates after second line treatments are considerably lower (20% to 50%), and response durations average 6 months.
Ovarian cancer is also highly curable in its earliest stages, but the overwhelming majority of patients are diagnosed in stages III and IV. Although responsive to chemotherapy, most patients with advanced ovarian cancer relapse and die of their disease. With the introduction of several neu cytotoxic agents (taxanes, vinorelbine, platinum derivatives), some responses are observed after second line therapy too, but cure in this situation remains an elusive goal.
Overexpression of the HER2/neu oncogene correlates with poor survival for breast and ovarian cancer patients and induces metastatic potential and chemoresistance of human cancer cells. Repression of HER2/neu suppresses the malignant phenotypes of HER2/neu-overexpressing cancer cells, suggesting that HER2/neu oncogene is an excellent target for the development of novel therapeutic agents against the HER2/neu-overexpressing cancer cells. Thus methods and compositions that repress HER2/neu transcription in HER2/neu-overexpressing human breast and ovarian cancer cell lines, and suppress activated neu induced transformation would be of great therapeutic value in the treatment of these diseases. PEA3, as a transcription factor, targets HER2/neu gene by repressing its expression, thus it has a great potential to be used as a therapeutic strategy of these neu-mediated cancer types.
In 10-20% of the HER2 overexpressing breast tumors, some gastric and virtually all HER2+ lung cancers HER2 mRNA and protein overexpression occur in the absence of increased gene copy number thus suggesting that HER2 there may be some aberration in transcriptional regulation that plays a fundamental role in the development of these diseased states.
Coexpression of PEA3 and HER2/neu stimulated PEA3-dependent reporter gene expression to a much greater extent than did either protein alone suggesting that HER2/neu upregulates the transcriptional activity of PEA3 (O""Hagan and Hassell, 1998). Overexpression of Rap1a, a ras-related protein capable of antagonizing ras function, completely inhibited the ability of HER2/neu to stimulate PEA3-dependent gene expression. Ras is known to stimulate at least two mitogen-activated protein kinase (MAPK) cascades, the extracellular-regulated kinase (ERK) cascade and the stress-activated kinase (SAPK) or Jun kinase (JNK) cascade. HER2/neu also activated both ERKs and SAPKs/JNKs in a Ras-dependent fashion. O""Hagan and Hassell, 1998 found that dominant-inhibitory mutants in either the ERK or SAPK/JNK cascades partially inhibited HER2/neu activation of PEA3-dependent gene expression, suggesting that HER2/neu regulates PEA3 activity through two different Ras-dependent MAPK pathways.
It has been shown that neu transcription can be enhanced by a variety of growth regulatory agents such as phorbol esters, epidermal growth factor and dibutyryl cAMP. Studies with neu promoters have identified cis and trans acting elements that may be involved in the regulation of neu transcription. Many DNA-binding trans-acting proteins are capable of stimulating DNA replication as well as gene transcription. The identification of a specific neu transactivator potentially leads to a molecular understanding of the development of neu gene amplification.
Recent studies have shown that there is an ETS response element that is conserved within a DNase I hypersensitive site in the proximal HER2 promoter region. This study concluded that ETS factors direct the overexpression of many gene products critical for human breast tumorogenesis. In yet another study, it was demonstrated that PEA3, a newly identified member of the ETS family is over-expressed in mouse metastatic mammary adenocarcinoma.
In other, contradictory studies when ETS-1 was ectopically expressed in two different highly tumorigenic human colon cancer cell lines it reversed the transformed phenotype and tumorigenicity in a dose dependent manner (Suzuki et al., 1995). A further study raised the question of suppresser activity for some ETS-1 products in T-cell acute lymphoblastic leukemias.
Hence it appears that there is much confusion regarding the putative role of the ETS family of transcription regulators.
The present invention generally relates to methods for repressing or preventing transformation in a cell, the method comprising contacting the cell with a polyomavirus enhancer activator 3 (PEA3), including but not limited to human PEA3 (hPEA3) or murine PEA3 (mPEA3) in an amount effective to inhibit a transformed phenotype. Inhibition of transformation may be indicated by a reduction in a transforming, tumorigenic or metastatic potential of a cell. Such cells may be in cell culture. More preferably, the cell in which transformation is to be repressed are cells in a living organism, for example a human. The inhibition of such transformation has great utility in the prevention and treatment of such transformation-driven events such as cancer, tumorigenesis, and metastasis.
Herein, the terms xe2x80x9cPEA3 gene productxe2x80x9d and xe2x80x9cPEA3xe2x80x9d refer to proteins having amino acid sequences which are substantially identical to human PEA3 (hPEA3) or murine PEA3 (mPEA3) or which are biologically active in that they are capable of cross-reacting with anti-PEA3 antibody raised against PEA3. xe2x80x9cPEA3 gene productxe2x80x9d and xe2x80x9cPEA3xe2x80x9d refer to proteins having amino acid sequences which are substantially identical to human PEA3 (hPEA3) or murine PEA3 (mPEA3) amino acid sequence and which are biologically active in that they are capable of binding to ETS binding sites or cross-reacting with anti-hPEA3 or anti-mPEA3 antibodies raised against hPEA3 or mPEA3, respectively. xe2x80x9cPEA3 gene productxe2x80x9d also includes analogs of hPEA3 or mPEA3 molecules which exhibit at least some biological activity in common with hPEA3 or mPEA3, respectively.
Herein, the term xe2x80x9cPEA3 genexe2x80x9d refers to any DNA sequence that is substantially identical to a DNA sequence encoding an hPEA3 or mPEA3 gene product as defined above. The term also refers to RNA, or antisense sequences compatible with such DNA sequences. A xe2x80x9cPEA3 genexe2x80x9d may also comprise any combination of associated control sequences.
As used in this specification and the appended claims, the singular forms xe2x80x9caxe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d generally mean xe2x80x9cat least onexe2x80x9d, xe2x80x9cone or morexe2x80x9d, and other plural references unless the context clearly dictates otherwise. Thus, for example, references to xe2x80x9ca cellxe2x80x9d, xe2x80x9ca polypeptidexe2x80x9d and xe2x80x9ca sequencexe2x80x9d include mixtures of cells, one or more polypeptides and a plurality of sequences of the type described; and reference to xe2x80x9cPEA3xe2x80x9d includes different species of such PEA3 and so forth.
PEA3 is a polypeptide that may be contacted with or introduced to a cell through any of a variety of manners known to those of skill. The PEA3 polypeptide may be introduced through direct introduction of a human PEA polypeptide to a cell. In this case, the PEA3 polypeptide may be obtained through any method known in the art, although it is anticipated that in vitro expression of the PEA3 polypeptide in a cell culture system may be a preferred manner of obtaining PEA3.
PEA3 may also be introduced to a cell via the introduction of a nucleic acid that encodes the PEA3 polypeptide to the cell. For example, RNA or DNA encoding PEA3 may be introduced to the cell by any manner known in the art.
In certain preferred embodiments, the PEA3 is introduced into the cell through the introduction of a DNA segment which encodes PEA3. In some such embodiments it is envisioned that the DNA segments will further comprises the PEA3 gene operatively linked to its associated control sequences. For example, the PEA3 gene may be operatively linked to a suitable promoter and a suitable terminator sequence. The construction of such gene/control sequence DNA constructs is well-known within the art. In particular embodiments the promoter is selected from the group consisting of CMV, SV40 IE and RSV LTR. In certain embodiments for introduction, the DNA segment may be located on a vector, for example, a plasmid vector or a viral vector. The viral vector may be, for example, a retroviral vector or an adenoviral vector. Such a DNA segment may be used in a variety of methods related to the invention. The vector may be used to deliver a PEA3 gene to a cell in one of the gene-therapy embodiments of the invention. Also, such vectors can be used to transform cultured cells, and such cultured cells could be used, inter alia, for the expression of PEA3 in vitro.
In some aspects of the invention PEA3 is used to inhibit oncogene-mediated transformation. Particular forms of oncogene-mediated transformation against which PEA3 is effective are exemplified by, but not limited to, neu or ras oncogene-mediated transformation. Some preferred embodiments of the present invention take advantage of the discovery disclosed herein that PEA3 binds to a region in the HER2/neu promoter. In more preferred embodiments the PEA3 binds to a region on the HER2/neu promoter that comprises a sequence of AGGAAG.
In certain aspects of the invention, the PEA3 polypeptide or encoding nucleic acid is complexed with a liposome for introduction to a cell. In some embodiments, the liposome comprises one or more of DOTMA, DOPE, or DC-Chol. In some specific embodiments, the liposome comprises DC-Chol. In other embodiments the liposome comprises DC-Chol and DOPE.
In particular embodiments the PEA3 is introduced into a cell that is a human cell. In many embodiments the cell is a tumor cell. In certain exemplary embodiments the tumor cell is a breast tumor cell or an ovarian tumor cell.
The present invention further provides methods to suppress the growth of an oncogene-mediated tumor in a mammal, the method comprising administering to said tumor a composition comprising PEA3, wherein said administration results in a decrease in the growth rate of said tumor. In some preferred embodiments, the introduction of PEA3 is affected by introduction a nucleic acid encoding PEA3 operatively linked to a promoter wherein the production of the PEA3 results in a decrease in the growth rate of said tumor. In particular embodiments the oncogene-mediated tumor is exemplified by a neu-mediated tumor or a ras-mediated tumor. In some preferred aspects of the present invention, the PEA3 polypeptide or encoding nucleic acid is administered in a liposomal complex.
The PEA3 gene products and nucleic acids of the present invention may also be introduced using any suitable method. A xe2x80x9csuitable methodxe2x80x9d of introduction is one that places a PEA3 gene product in a position to inhibit the transformation of a cell. For example, injection, oral, and inhalation methods may be employed, with the skill artisan being able to determine an appropriate method of introduction for a given circumstance. In some preferred embodiments, injection will be used. This injection may be intravenous, intraperitoneal, intramuscular, subcutaneous, intratumoral, intrapleural, or of any other appropriate form.
In certain other aspects of the present invention there are provided therapeutic kits comprising in suitable container, a pharmaceutical formulation of a PEA3 gene product or a nucleic acid encoding a PEA3 gene product. Such a kit may further comprise a pharmaceutical formulation of a therapeutic polypeptide, nucleic acid encoding a therapeutic polypeptide, or chemotherapeutic agent.
In some embodiments of the present invention, the discovery that PEA3 is able to inhibit transformation will be used in combination with other anti-transformation/anti-cancer therapies. These other therapies may be known at the time of this application, or may become apparent after the date of this application. PEA3 may be used in combination with other therapeutic polypeptides, nucleic acid encoding other therapeutic polypeptides, or chemotherapeutic agents. For example, PEA3 may be used in conjunction with other known anti-cancer polypeptides, such as P53. PEA3 may be used in conjunction with any known transformation or disease inhibitor. PEA3 may be used with other gene-therapy regimes. PEA3 may be used with any suitable chemotherapeutic drug.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.